This is a application to seize the opportunity of the co-existence of two paradigm-shifting technologies at the University of Washington and, by adding a third, bring them together to address one of the major challenges in biomedical research - quantitative descriptions of intricate developmental, physiological and pathological relationships in expression patterns between multiple cell types in complex tissues. Dr. Morris is a biochemist/molecular biologist who has developed the "RiboTag mouse", which enables isolation and analysis of ribosome-associated transcriptomes of individual cell types in a tissue without prior cell separations. Dr. Shendure is a genome scientist who pioneered the development of one of the first next-generation sequencing platforms and has extensive expertise in developing new technologies for second-generation sequencing and analysis of large-scale genomic data, including RNA-Seq. The new hybrid technology - RiboTag-seq - will not only provide highly quantitative measurements of transcript levels in individual cell types, but will yield robust estimates of the translation of individual proteins by mapping the numbers and positions of ribosomes engaged with transcriptomes of interest. Drs. Morris and Shendure are teaming with Dr. Nelson, who is at the Fred Hutchinson Cancer Research Center and an expert in tumor-microenvironment interaction, to apply RiboTag-seq to analyzing reciprocal interactions between a tumor and its adjacent cellular milieu, i.e. the tumor microenvironment. In this instance, prostatic epithelial and stromal transcriptomes will be analyzed during the course of carcinogenesis in a well-defined mouse model. The results from RiboTag-seq will be compared with those from expression arrays performed in parallel and the strengths and weaknesses of the two approaches evaluated. The outcome of this analysis of prostate cancer in situ will lead to evaluation of two commonly used models of neoplasia-tumor xenografts in nude mice and co-culture in vitro of tumor cells and stromal fibroblasts. This project will be deployed immediately upon funding and will create 4 new full-time research positions and a part-time student position. When completed, the project will provide the basis for more expansive studies of tumor microenvironment and, more significantly, allow extension of the RiboTag-seq technology to examination of any other complex tissue of interest. PUBLIC HEALTH RELEVANCE: One of the major challenges in biomedical research is the analysis of intricate developmental, physiological and pathological relationships in gene expression between multiple cell types in complex tissues. This project brings together a biochemist/molecular biologist, a tumor biologist and a genome scientist, along with a paradigm-shifting set of experimental tools that are currently available at the University of Washington, to address the reciprocal interactions between a tumor and its surrounding cellular milieu - the tumor microenvironment. Besides creating access to burning questions in tumor biology, the overriding importance of this project is that it will provide entire through the RiboTag-seq methodology to studies of any other complex tissue of interest.